differential role of cyclooxygenase 1 and 2 isoforms in...

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Differential role of cyclooxygenase 1 and 2 isoforms in the modulation of

colonic neuromuscular function in experimental inflammation

Matteo Fornai, Corrado Blandizzi, Luca Antonioli, Rocchina Colucci, Nunzia Bernardini,

Cristina Segnani, Fabrizio De Ponti, Mario Del Tacca

Division of Pharmacology and Chemotherapy, Department of Internal Medicine (M.F, C.B.,

L.A., R.C., M.D.T.), Department of Human Morphology and Applied Biology, Section of

Histology and General Embryology (N.B., C.S.), University of Pisa, Pisa, Italy, Department

of Pharmacology (F.D.P.), University of Bologna, Bologna, Italy

JPET Fast Forward. Published on February 10, 2006 as DOI:10.1124/jpet.105.098350

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on February 10, 2006 as DOI: 10.1124/jpet.105.098350

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Running Title: COX isoforms and colonic motility

Corresponding Author:

Prof. Corrado Blandizzi, M.D.

Divisione di Farmacologia e Chemioterapia, Dipartimento di Medicina Interna

Università di Pisa

Via Roma, 55, 56126 Pisa, Italy

Tel.: +39-050-830148; Fax: +39-050-562020; e-mail: [email protected]

Number of text pages: 27

Number of tables: 1

Number of figures: 7

Number of references: 28

Number of words in the abstract: 250

Number of words in the introduction: 498

Number of words in the discussion: 1028

Abbreviations: COX, cyclooxygenase; DAB, 3,3’-diaminobenzidine tetrahydrochloride;

DNBS, 2,4-dinitrobenzenesulfonic acid; dNTP, deoxynucleotide triphosphate; L-NAME, Nω-

nitro-L-arginine methylester; NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible

nitric oxide synthase; PBS, phosphate buffered saline; ROS, reactive oxygen species; RT-

PCR, reverse transcription-polymerase chain reaction; SEM, standard error of mean; SMT, S-

methylisothiourea; SOD, superoxide dismutase; TES, transmural electrical stimulation.

Recommended section assignment: Gastrointestinal, Hepatic, Pulmonary, and Renal


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ABSTRACT

This study examines the role played by cyclooxygenase isoforms (COX-1 and COX-2) in the

regulation of colonic neuromuscular function in normal rats and after induction of colitis by

2,4-dinitrobenzenesulfonic acid (DNBS). The expression of COX-1 and COX-2 in the colonic

neuromuscular layer was assessed by RT-PCR and immunohistochemistry. The effects of

COX inhibitors on in vitro motility were evaluated by studying electrically induced and

carbachol-induced contractions of the longitudinal muscle. Both COX isoforms were

constitutively expressed in normal colon; COX-2 was upregulated in the presence of colitis.

In normal and inflamed colon, both COX isoforms were mainly localized in neurones of

myenteric ganglia. In the normal colon, indomethacin (COX-1/COX-2 inhibitor), SC-560

(COX-1 inhibitor) or DFU (COX-2 inhibitor) enhanced atropine-sensitive electrically evoked

contractions. The most prominent effects were observed with indomethacin or SC-560 plus

DFU. In the inflamed colon, SC-560 lost its effect, whereas indomethacin and DFU

maintained their enhancing actions. These results were more evident after blockade of non-

cholinergic pathways. In rats with colitis, in vivo treatment with superoxide dismutase or S-

methylisothiourea (inhibitor of inducible nitric oxide synthase) restored the enhancing motor

effect of SC-560. COX inhibitors had no effect on carbachol-induced contractions in normal

or DNBS-treated rats. In conclusion, in the normal colon both COX isoforms act at neuronal

level to modulate the contractile activity driven by excitatory cholinergic pathways. In the

presence of inflammation, COX-1 activity is hampered by oxidative stress, and COX-2 seems

to play a predominant role in maintaining an inhibitory control of colonic neuromuscular

function.


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Introduction

Since the discovery of two cyclooxygenase (COX) isoforms (COX-1, COX-2), efforts

have been made to examine the distribution and functional roles played by these enzymes in

the gastrointestinal tract. The mucosal layer of normal gut expresses high levels of COX-1

and low levels of COX-2 (Fornai et al., 2005a; Wallace and Devchand, 2005), whereas in the

presence of ulcer, inflammatory or neoplastic diseases, COX-2 expression can be upregulated.

In these settings, COX isoforms may play differential pathophysiological roles, such as the

involvement of COX-1 and COX-2 in the development of gastrointestinal damage induced by

non-steroidal anti-inflammatory drugs (NSAIDs) (Tanaka et al., 2002), and the carcinogenesis

and tumour cell proliferation promoted by COX-2-derived prostanoids (Subbaramaiah and

Dannenberg, 2003). By contrast, the pattern of COX isoform expression in gut neuromuscular

layers remains to be clarified. Previous reports described a significant COX-2 expression in

colonic myenteric plexus and myocytes of various species, including humans, under normal

conditions (Porcher et al., 2004; Fornai et al., 2005b), whereas other authors reported a scarce

or absent expression (Roberts et al., 2001; Schwarz et al., 2001).

Intestinal inflammation is associated with alterations of gut motility and may contribute

to the development of digestive symptoms (Collins, 1996). The mechanisms underlying

dysmotility are still uncertain, but altered functions of myenteric nerves and smooth muscle

have been observed (Sharkey and Kroese, 2001). Previous studies reported that COX

pathways are involved in the modulation of normal gastrointestinal motility (Porcher et al.,

2002; Porcher et al., 2004; Fornai et al., 2005b), and that in the presence of intestinal

inflammatory reactions COX-2 might contribute to the pathophysiology of related motor

alterations (Schwarz et al., 2001; Linden et al., 2004). For instance, experimental ileus in rats,

evoked by intestinal manipulation, was associated with enteric COX-2 induction, and


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treatments with selective COX-2 inhibitors significantly improved the intestinal contractile

activity, both in vivo and in vitro (Schwarz et al., 2001). Moreover, experimental colitis

induced by trinitrobenzenesulfonic acid in guinea-pigs is characterized by increased COX-2

expression in the colonic wall, and the products of this enzyme seem to be responsible for an

enhanced excitability of myenteric AH neurones. In this model, the inhibition of COX-2, but

not COX-1, restored the normal electrical properties of AH neurones, whereas the application

of prostaglandin E2 to inflamed colonic preparations decreased the afterhyperpolarization of

AH neurones and slowed their accommodation rate (Manning et al., 2002; Linden et al.,

2004). However, there is still uncertainty on the role of COX isoforms in motor alterations

associated with chronic intestinal inflammation and on the hypothesis that COX-derived

mediators may regulate differently gut motility under physiological or pathological conditions

(Costa, 2004).

The present study was designed to compare the role of COX isoforms in the control of

neuromuscular functions in normal rats and after the induction of colitis. For this purpose, we

examined the expression and localization of COX-1 and COX-2 in the colonic neuromuscular

layer, and the effects of COX blockade on in vitro colonic motor activity, using selective and

non-selective COX-inhibitors.


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Methods

Animals

Albino male Sprague-Dawley rats, 200-250 g body weight, were used throughout the

study. They were housed in temperature-controlled rooms, in a 12-h light-dark cycle at 22-24

°C and 50-60% humidity. Their care and handling were in accordance with the provision of

the European Union Council Directive 86/609, recognized and adopted by the Italian

Government.

Induction and assessment of colitis

Colitis was induced in accordance with the method previously described by Barbara et

al. (2000). Briefly, during anaesthesia with diethyl ether, 30 mg of 2,4-

dinitrobenzenesulphonic acid (DNBS) in 0.25 ml of 50% ethanol were administered

intrarectally via a polyethylene PE-60 catheter inserted 8 cm proximal to the anus. Control

rats received 0.25 ml of saline. Animals underwent subsequent experimental procedures 6

days after DNBS administration, to allow a full development of histologically evident colonic

inflammation. At that time, the animals were euthanized, and the severity of intestinal

inflammation was evaluated macroscopically and histologically in accordance with the

criteria previously reported by Wallace & Keenan (1990), as modified by Barbara et al.

(2000). The macroscopic criteria were based on the following: presence of adhesions between

colon and other intra-abdominal organs; consistency of colonic faecal material (indirect

marker of diarrhoea); thickening of colonic wall; presence and extension of hyperaemia and

macroscopic mucosal damage (assessed with the aid of a ruler). Microscopic criteria were

assessed by light microscopy on haematoxylin- and eosin-stained sections obtained from

whole-gut specimens, taken from a region of inflamed colon immediately adjacent to the

gross macroscopic damage and fixed in cold 4% neutral formalin diluted in phosphate

buffered saline (PBS). Histological criteria included: degree of mucosal architecture changes;


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cellular infiltration; external muscle thickening; presence of crypt abscess and goblet cell

depletion. All parameters of macroscopic and histological damage were recorded and scored

for each rat by two observers blinded to the treatment.

Reverse transcription-polymerase chain reaction

Expression of mRNA coding for COX isoforms was assessed by reverse transcription-

polymerase chain reaction (RT-PCR). The analysis was performed on colonic specimens

excised as reported above, subjected to mucosa and submucosa removal, snap-frozen in liquid

nitrogen, and stored at -80°C. At the time of extraction, tissue samples were disrupted with

cold glass pestles and total RNA was isolated by Trizol (Life Technologies, Carlsbad, CA,

U.S.A.) and chloroform. Total RNA (1 µg) served as template for cDNA synthesis in a

reaction using 2 µl random hexamers (0.5 µg/µl) with 200 U of MMLV-reverse transcriptase

in a buffer containing 500 µM deoxynucleotide triphosphate mixture (dNTP) and 10 mM

dithiothreitol. cDNA samples were subjected to PCR in the presence of primers based on

cloned rat COX isoforms (Tanaka et al., 2002). PCR, consisting of 5 µl of RT products, Taq

polymerase 2.5 U, dNTP 100 µM and primers 0.5 µM, was carried out by a PCR-Express

thermocycler (Hybaid, Ashford, Middlesex, U.K.). After 3 min at 94°C, the cycle conditions

were 1 min at 94°C, 1 min at 55°C and 1 min at 72°C for 30 cycles, followed by 7 min at

72°C. Aliquots of RNA not subjected to RT were included in PCR reactions to verify the

absence of genomic DNA. The efficiency of RNA extraction, RT and PCR was evaluated by

primers for rat β-actin. PCR products were separated by 1.8% agarose gel electrophoresis in a

Tris buffer 40 mM containing 2 mM ethylenediamine tetraacetic acid, 20 mM acetic acid (pH

8), and stained with ethidium bromide. PCR products were then visualized by UV light and

subjected to densitometric analysis by Kodak Image Station program (Eastman Kodak Co.,

Rochester, NY, U.S.A.). The relative expression of target mRNA was normalized to that of β-

actin.


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Immunohistochemical analysis

Specimens of colonic tissue, excised and fixed as reported above, were dehydrated with

ethanol, treated with xylene and embedded in paraffin at 56°C. Serial sections (5-µm-thick)

were processed for immunostaining: slides were treated with 1% hydrogen peroxide in

methanol, microwaved in citrate buffer, and blocked with normal swine serum (1:20;

Dakopatts, Glostrup, Denmark). Sections were then incubated overnight at 4°C with the

following polyclonal primary antibodies: rabbit anti-COX-1 (1:200; Cayman Chemical

Company, code no. 160109, Ann Arbor, Michigan, U.S.A.); rabbit anti-COX-2 (1:3000;

Alexis Biochemicals, code no. ALX 210711, Lausen, Switzerland); rabbit anti-neurofilament

(1:4000; Chemicon, code no. AB 1987, Temecula, California, U.S.A.). Immunoglobulins

were diluted in PBS with 0.1% bovine serum albumin and 0.1% sodium azide. Sections were

washed with PBS and incubated with biotinylated immunoglobulins followed by peroxidase-

labeled streptavidin complex and 3,3’-diaminobenzidine tetrahydrochloride (DAB;

Dakopatts, Glostrup, Denmark) (Bernardini et al., 1999). Sections were counterstained with

haematoxylin. All reactions were carried out at room temperature in a humidified chamber

and PBS was used for washes, unless otherwise specified. Negative controls were obtained by

omitting primary antibodies or substituting the primary antibody with rabbit preimmune

serum. Specificity of COX immunoreacting staining was assessed by pre-adsorbing anti-

COX-1 and -COX-2 antibodies with COX-1 (Cayman Chemical Company, cod. no. CAY

360109) and COX-2 (Alexis Biochemicals, cod. no. ALX 153-063) blocking peptides,

respectively, at ten times the antibody concentrations for 24 h at 4°C. To test endogenous

peroxidases and avidin-binding activity, slides were incubated only with DAB or streptavidin-

peroxidase complex plus DAB, respectively.


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Recording of longitudinal muscle contractile activity

The contractile activity of colonic longitudinal smooth muscle was recorded as

previously described by Blandizzi et al. (2003). Specimens of colon, excised as reported

above, were placed into cold pre-oxygenated Krebs solution, opened along the mesenteric

insertion and subjected to removal of mucosal/submucosal layer. The specimens were then

cut along the longitudinal axis into strips of approximately 3 mm width and 20 mm length.

The preparations were set up in 10-ml organ baths containing Krebs solution at 37°C, bubbled

with 95% O2+5% CO2, connected to isotonic transducers (Basile, Comerio, Italy) under a

constant load of 1 g, and allowed to equilibrate for 30 min. Since there was no need to

discriminate between active and passive tension developed by intestinal muscle, isotonic

transducers were used to estimate changes in smooth muscle elongation under constant

tension in response to stimulation. Krebs solution had the following composition (mM): NaCl

113, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 11.5 (pH 7.4±0.1).

The contractile activity was recorded by a polygraph (Basile, Comerio, Italy). A pair of

coaxial platinum electrodes was positioned at distance of 10 mm from longitudinal axis of

each preparation to deliver transmural electrical stimulation (TES) by a BM-ST6 stimulator

(Biomedica Mangoni, Pisa, Italy). Stimuli were applied as 10-s single trains of square wave

pulses (0.5 ms, 30 mA, 10 Hz). Each preparation was repeatedly challenged with electrical

stimulations, and experiments started when reproducible responses were obtained (usually

after 2-3 stimulations).

In the first set of experiments, colonic preparations were exposed to indomethacin

(COX-1/COX-2 inhibitor, 1 µM), SC-560 (COX-1 inhibitor, 0.1 µM) or DFU (COX-2

inhibitor, 1 µM), for 30 min before TES. Preparations were incubated with test drugs along

two 15-min consecutive periods with an intervening washing. Drug concentrations were

selected on the basis of previous studies (Riendeau et al., 1997; Kato et al., 2001).


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The second set of experiments was designed to assay COX inhibitors on contractile

responses elicited by TES directed to cholinergic innervation. For this purpose, colonic

preparations were maintained in Krebs solution containing guanethidine (adrenergic blocker,

10 µM), Nω-nitro-L-arginine methylester (L-NAME, inhibitor of nitric oxide synthase, NOS,

100 µM), L-732,138 (NK1 receptor antagonist, 10 µM), GR-159897 (NK2 receptor antagonist,

1 µM) and SB-218795 (NK3 receptor antagonist, 1 µM), to prevent non-cholinergic motor

responses (Fornai et al., 2005b). Incubation of colonic strips with COX inhibitors before

challenge with TES was performed as reported above.

In the third series, COX inhibitors were assayed on cholinergic contractions elicited by

direct pharmacological activation of muscarinic receptors on smooth muscle cells.

Preparations were maintained in Krebs solution containing tetrodotoxin (1 µM), and

stimulated twice with carbachol (muscarinic receptor agonist, 1 µM). The first stimulation

was applied in the absence of other drugs, while the second one was applied after 30-min

incubation with COX inhibitors, as reported above.

In a fourth set of experiments, the effects of COX inhibitors on TES-evoked motor

responses were assessed on colonic preparations obtained from control or DNBS-treated rats

treated daily with superoxide dismutase (SOD, 7 mg/kg/day s.c.) (Segui et al., 2004) or S-

methylisothiourea (SMT, 14 mg/kg/day s.c.), a selective inhibitor of inducible NOS (iNOS)

(Afulukwe et al., 2000), for 6 consecutive days starting 1 h before the induction of colitis.

Drugs and reagents

Indomethacin, SC-560 ([5-(4-clorophenyl)-1-(4-metoxyphenyl)-3-trifluoromethyl-

pirazole), atropine sulphate, hexamethonium bromide, Nω-nitro-L-arginine methylester,

carbachol hydrochloride, guanethidine, superoxide dismutase, S-methylisothiourea (Sigma

Chemical, St. Louis, MO, U.S.A.); DFU [3-(3-fluorophenyl)-4-(4-methanesulfonyl)-5,5-

dimethyl-5H-furan-2-one] (kindly provided by Merck Research Laboratories, Rahway, NJ,


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U.S.A.); L-732,138 (N-acetyl-L-tryptophan 3,5-bis(trifluoromethyl)benzyl ester), GR-159897

(5-fluoro-3-[2-[4-methoxy-4-[[(R)-phenylsulphinyl]methyl]-1-piperidinyl]ethyl]-1H-indole),

SB-218795 ((R)-[[2-phenyl-4-quinolinyl)carbonyl]amino]-methyl ester benzeneacetic acid),

tetrodotoxin (Tocris Cookson, Bristol, U.K.); random hexamers, MMLV-reverse

transcriptase, Taq polymerase, dNTP mixture (Promega, Madison, WI, U.S.A.). COX

inhibitors were dissolved in dimethylsulphoxide and further dilutions were made with saline

solution. Dimethylsulphoxide concentration in organ bath never exceeded 0.5%.

Statistical analysis

Results are given as mean standard ± error of mean (SEM). The significance of

differences was evaluated on raw data, prior to percentage normalization, by Student t test for

unpaired data or one way analysis of variance followed by post hoc analysis with Student-

Newman-Keuls test, and P<0.05 was considered significant. Colonic preparations included in

each test group were obtained from distinct animals, and therefore the number of experiments

refers also to the number of animals assigned to each group. Calculations were performed by

commercial software (GraphPad Prism™, version 3.0 from GraphPad Software Inc., San

Diego, CA, U.S.A.).


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Results

Assessment of colitis

Six days after DNBS treatment, the distal colon appeared thickened and ulcerated with

evident areas of transmural inflammation. Adhesions were often present and the bowel was

occasionally dilated. Histologically, colitis was evident as granulocyte infiltration extending

throughout the mucosa and submucosa, sometimes involving the muscular layer. More than

five-fold increase in both macroscopic and microscopic damage score was estimated in

DNBS-treated animals in comparison with normal rats. In rats with colitis, treated with SOD

or SMT, both macroscopic and microscopic damage scores were significantly decreased,

although being still significantly greater than normal values (Table 1).

RT-PCR analysis

RT-PCR analysis revealed the expression of COX-1 and COX-2 in colonic

neuromuscular layers of both normal and DNBS-treated animals (Figure 1A). The

densitometric analysis, performed on amplified cDNA bands, indicated a significant increase

in the expression of COX-2 mRNA in the presence of colitis (Figure 1C), whereas no

appreciable changes in the expression of COX-1 were detected (Figure 1B).

Immunohistochemical analysis

Both COX-1 and COX-2 are constitutively expressed in the tunica muscularis of normal

rat colon (Figure 2A and C). In particular, COX-1 was found in neurons and glial cells of

myenteric ganglia as well as in few cells of muscular layer (Figure 2A and 3A), whereas little

COX-2 immunoreactivity was expressed only in myenteric neurons (Figure 2C and 3C).

Colonic neuromuscular tissues from animals with colitis showed different patterns of COX

isoform expression (Figure 2B, D and 3B, D) compared to normal rats. Myenteric ganglionic

cells and few smooth muscle cells expressed detectable amount of COX-1 (Figure 2B and

3B), which was similar to normal colonic tissues. By contrast, high COX-2 expression was


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found in the neuromuscular compartment of colonic specimens excised from animals with

colitis: intense COX-2 immunoreactivity was detected in myenteric ganglionic cells and

muscular layers (Figure 2D and 3D). In the latter case, COX-2 was mainly localized at level

of longitudinal muscle cell nuclei (Figure 2D), in accordance with previously reported COX-2

nuclear labelling (Maihofner et al., 2000). Neurones of myenteric ganglia were localized by

means of immunoreactivity to neurofilament both in normal (Figure 2E) and inflamed colon

(Figure 2F). Pre-adsorption of primary antibodies with the respective blocking peptides

completely abolished any specific immunoreactivity (data not shown).

Effects of COX inhibitors on longitudinal smooth muscle activity

During the equilibration period, most colonic preparations, obtained from normal or

DNBS-treated rats, showed rapid and low in amplitude spontaneous activity, which remained

stable throughout the experiment. TES-induced responses consisted of fast phasic

contractions often followed by aftercontractions of variable amplitude (Figure 4). The use of

isotonic transducers allowed to record electrically-evoked contractions as changes in smooth

muscle elongation under constant tension, thus minimizing possible inflammation-induced

variations of intrinsic contractile activity. Accordingly, control responses of normal tissues

did not differ significantly from those observed in colonic preparations from DNBS-treated

rats. Pre-incubation with atropine (1 µM) inhibited phasic contractions, or converted them

into relaxations, and only aftercontractions were evident. Tetrodotoxin (1 µM) abolished the

contractile responses evoked by TES (not shown).

Incubation of colonic preparations from normal or DNBS-treated animals with

indomethacin, SC-560 or DFU was not associated with significant changes in spontaneous

motor activity. In normal colonic tissues, indomethacin significantly enhanced contractions

evoked by TES (+64%, 1 µM) (Figure 4 and 5A). SC-560 (0.1 µM) or DFU (1 µM) mimicked

this enhancing action, but they were less effective than indomethacin (+41% and +36%)


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(Figure 4 and 5A). After combined incubation with SC-560 plus DFU, TES elicited

contractile responses with amplitude (+61%) comparable to that observed with indomethacin

(Figure 5A). In colonic preparations from rats with colitis, indomethacin (1 µM) or DFU (1

µM) evoked significant and similar increments of TES-induced contractions (+32% and

+34%, respectively) (Figure 4 and 5B), whereas SC-560 (0.1 µM) was without effect (Figure

4 and 5B). After co-incubation of colonic tissues with SC-560 plus DFU the enhancement of

TES-induced contractions was similar to that observed in the presence of indomethacin or

DFU alone (+39%) (Figure 5B).

In the presence of guanethidine, L-NAME and NK receptor antagonists, TES evoked

contractions of colonic preparations, obtained from normal or DNBS-treated rats, which were

completely prevented, or markedly reduced, by atropine (1 µM, not shown). Under these

conditions, TES-induced motor responses were suppressed by tetrodotoxin (1 µM) and

unaffected by hexamethonium (10 µM). In normal preparations, indomethacin (1 µM)

significantly enhanced cholinergic responses elicited by TES (+94%) (Figure 5C). SC-560

(0.1 µM) or DFU (1 µM) mimicked this excitatory effect, although they were less effective

(+58% and +61%) (Figure 5C). Incubation with SC-560 plus DFU was followed by a

potentiation of TES-induced contractions (+97%) similar to that achieved with indomethacin

alone (Figure 5C). In colonic tissues isolated from rats with colitis, indomethacin (1 µM) or

DFU (1 µM) significantly increased TES-induced contractions, and acted with similar

efficacy (+55% and +49%, respectively), whereas SC-560 (0.1 µM) did not exert any

significant effect (Figure 5D). Following co-incubation of colonic tissues with SC-560 plus

DFU, TES-evoked contractions were enhanced to a similar extent to that observed with

indomethacin or DFU alone (+64%) (Figure 5D).


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Exposure of colonic preparations, from normal or DNBS-treated animals, to carbachol

(1 µM) resulted in phasic contractions (Figure 6) sensitive to atropine. Under these

conditions, carbachol-induced responses were not affected by indomethacin (1 µM) either in

the absence or in the presence of colitis (Figure 6). Analogously, SC-560 (0.1 µM), DFU (1

µM) or SC-560 plus DFU did not modify the motor responses elicited by carbachol (Figure

6).

When colonic preparations, isolated from animals treated with DNBS plus SOD, were

exposed to indomethacin (1 µM), the contractile responses evoked by TES were significantly

increased (+66%) (Figure 7A). Under these conditions, SC-560 (0.1 µM) or DFU (1 µM)

significantly enhanced TES-evoked motor activity, although they were less effective than

indomethacin (+28% and +46%, respectively) (Figure 7A). Co-incubation of colonic tissues

with SC-560 plus DFU resulted in increments of TES-induced contractions similar to those

observed in the presence of indomethacin alone (+69%) (Figure 7A). In colonic tissues from

rats with colitis subjected to in vivo iNOS blockade by SMT, COX inhibitors enhanced TES-

induced contractions, with response patterns similar to those recorded after treatment of

animals with DNBS plus SOD (Figure 7B). Incubation of colonic tissues, obtained from

normal rats treated with SOD or SMT, with COX inhibitors, resulted in enhancements of

TES-induced contractions which did not differ significantly from those observed in the

absence of SOD or SMT.


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Discussion

This study provides evidence that: 1) both COX isoforms are constitutively expressed in

colonic neuromuscular layers in the rat and contribute to the modulation of cholinergic motor

activity acting at neuronal level; 2) in the presence of colitis, the COX-2 isoform plays a

predominant role in this regulatory function.

Our molecular and morphological analysis clearly demonstrated expression of both

COX-1 and COX-2 in the neuromuscular layer of normal rat colon and up-regulation of

COX-2 expression in the inflamed colon. In particular, immunohistochemistry indicated a

predominant localization of both COX isoforms in neurons of myenteric ganglia and an

increased immunostaining of COX-2 at the same sites in the presence of colitis. These

findings add evidence to the concept that COX-2 can be expressed constitutively in the

normal gut (as we and others have previously reported in human and mouse gut; Fornai et al.,

2005b; Porcher et al., 2002; Porcher et al., 2004) and could play a role in the control of

myenteric nerve function during intestinal inflammation. In addition, Roberts et al. (2001)

demonstrated increased COX-2 immunoreactivity in colonic myenteric neurons of patients

with ulcerative colitis or Crohn’s disease, and Schwarz et al. (2001) found enhancement of

COX-2 immunostaining in neurones and smooth muscle cells from jejunal tissues of rats

undergoing intestinal manipulation (an experimental model of postoperative ileus).

The pharmacological blockade of COX isoforms in normal colonic preparations

significantly increased electrically induced contractions, especially after pharmacological

ablation of non-cholinergic nerve pathways. Electrically evoked cholinergic contractions were

enhanced after exposure to either SC-560 (COX-1 inhibitor) or DFU (COX-2 inhibitor). Even

more pronounced effects (of similar magnitude) were recorded with the non selective

inhibitor indomethacin and with combined blockade of both COX isoforms by SC-560 plus

DFU.


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The present findings strengthen the notion that COX-derived products can modulate

enteric cholinergic neurotransmission in the normal gut. De Backer et al. (2003) reported that

indomethacin enhances the electrically induced acetylcholine release in muscle strips of pig

stomach, suggesting inhibition by prostanoids of intramural cholinergic neurones.

Furthermore, we previously observed that selective and non selective COX inhibitors

potentiate electrically induced cholinergic contractions of smooth muscle strips prepared from

human distal colon (Fornai et al., 2005b). In contrast with findings in humans, pigs and rats,

experiments performed on guinea-pig intestine indicated that the electrically or nicotine-

induced acetylcholine release and related cholinergic contractions are enhanced by application

of exogenous prostaglandins and inhibited by indomethacin, suggesting that in this species

endogenous prostanoids mediate excitation of cholinergic enteric pathways (Takeuchi et al.,

1991). Therefore, the effects of prostanoids and COX inhibitors on gut motility depend on

species, gut region and muscular layer.

Our observation that TES-, but not carbachol-induced contractions were affected by

COX-blockade indicates that the modulating control of COX pathways on cholinergic motor

activity occurs at neuronal rather than at muscular sites. These functional data are consistent

with our molecular analysis showing that both COX-1 and COX-2 were detected

predominantly in neurones of myenteric ganglia, with no appreciable immunostaining in the

muscular layers. Thus, it is conceivable that prostanoids produced by COX-1 and COX-2,

both located in myenteric neurones, inhibit colonic cholinergic neurotransmission. In line

with this proposal, preliminary in vivo experiments performed in our laboratory indicated that

colonic transit in normal animals was enhanced by either COX-1 or COX-2 blockade,

whereas COX-1 inhibition was without effects in rats with colitis (Del Tacca et al.,

unpublished data).


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In colonic preparations obtained from rats with colitis, SC-560 lost its enhancing effect

on electrically induced cholinergic contractions, whereas increments of similar degree were

observed upon application of indomethacin, DFU or indomethacin plus DFU. The modulating

function of COX-2 was likely to occur still at neuronal level, as neither indomethacin nor

DFU affected carbachol-induced contractions.

An intriguing finding in rats with colitis is that the functional data are not fully

consistent with the patterns of COX isoform localization: while expression of COX-2

increased, the motor enhancing effects resulting from its pharmacological inhibition remained

nearly unchanged. On the other hand, COX-1 expression did not vary, but its blockade was

without effects on the evoked cholinergic contractions. To get further insight into this aspect,

it is noteworthy that gut inflammation is associated with increased production of reactive

oxygen species (ROS), such as superoxide anion radicals (O2-) and peroxynitrite anions

(ONOO-), known to induce oxidative tissue injury (Dijkstra et al., 1998), and that

peroxynitrite anions, generated from the reaction between nitric oxide (NO) and superoxide

anions, can downregulate the catalytic activities of COX enzymes, especially COX-1

(Fujimoto et al., 2004). Accordingly, antioxidants were reported to ameliorate experimental

colitis (Segui et al., 2004; Oz et al., 2005). Thus, it is conceivable that, in the presence of

colitis, increased production of peroxynitrite anions could functionally inactivate COX-1

activity, whereas COX-2 overexpression could remain functionally silent because of the

inhibition exerted by reactive oxygen species. To test this hypothesis, we performed

functional experiments on colonic preparations obtained from rats with colitis treated with

SOD (the enzyme responsible for O2- inactivation) or SMT (a selective inhibitor of iNOS, the

main source of NO in inflamed tissues). Evidence was thus obtained that the in vivo

antioxidant treatment with SOD or SMT could prevent COX-1 inactivation. Indeed, under


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19

these conditions, SC-560 almost completely recovered its ability to enhance the electrically

evoked contractions of colonic longitudinal muscle strips.

Taking together our molecular and pharmacological findings, it appears that, in the

presence of inflammation, changes in the control of colonic motility by COX pathways reflect

mainly impaired function of these enzymes because of increased oxidative stress. Thus, as

postulated by Costa (2004), COX-derived mediators do not seem to play distinct

physiological and pathological roles, at least in the case of gut motility, and the induction of

COX-2 expression by intestinal inflammation might be viewed as an attempt of endogenous

homeostatic mechanisms to preserve or restore the modulating actions of COX products.

In conclusion, this study indicates that both COX-1 and COX-2 are constitutively

expressed in the neuromuscular layer of normal rat colon, where they are mainly localized in

neurones of myenteric ganglia and contribute to inhibition of excitatory cholinergic pathways.

In the presence of colitis, the COX-2 isoform seems to play a predominant role in driving this

modulating action.


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Footnotes

The present work was supported by a grant from the Italian Ministry of Education, University

and Research (PRIN-COFIN 2002, Project number 2002052573-003).


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Figure legends

Figure 1. Reverse transcription-polymerase chain reaction analysis of COX-1, COX-2 and β-

actin mRNA in the muscular layer of distal colon obtained from rats under normal conditions

or with DNBS-induced colitis. (A) Three representative agarose gels, referring to the

amplification of COX-1, COX-2 and β-actin cDNAs. (B, C) Column graphs referring to the

densitometric analysis of COX-1 and COX-2 cDNAs bands normalised to the expression of

β-actin. Each column represents the mean value ± SEM obtained from 6 experiments.

*P<0.05: significant difference vs normal values.

Figure 2. Immunohistochemical detection of COX-1 (A, B), COX-2 (C, D) and

neurofilament (NFL; E, F) performed on adjacent sections of colon from rats under normal

conditions (A, C, E, G) or with DNBS-induced colitis (B, D, F, H). Circular (cm) and

longitudinal (lm) smooth muscle. Neurons of myenteric ganglia (arrowheads). (A) Normal

colonic neuromuscular tissue shows detectable amount of COX-1 in neurons (arrowheads)

and glial cells of myenteric ganglia as well as in few smooth muscle cells of circular and

longitudinal muscular layers. (B) Rats with colitis show no appreciable variation of COX-1

expression in colonic neuromuscular structures compared to normal rats. (C) COX-2 is

weakly expressed in myenteric neurons (arrowheads) of colon from normal animals. (D)

COX-2 staining is enhanced in colonic neuromuscular structures of rats with colitis: COX-2 is

strongly expressed both in myenteric ganglia (arrowheads) and tunica muscularis mainly at

level of longitudinal layer. (E, F) Neurons (arrowheads) are identified within myenteric

neurons of colon from normal (E) or DNBS-treated (F) rats by anti-neurofilament (NFL)

immunoreaction. (G, H) No specific immunoreaction is observed in serial sections of

specimens from normal (G) or DNBS-treated (H) animals, incubated with preimmune rabbit

serum.


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Figure 3. Higher magnification of myenteric ganglia represented in boxed areas of Figure 2:

COX-1 (A, B) and COX-2 (C, D) were detected in colon from normal (A, C) and DNBS-

treated (B, D) rats. Neurons of myenteric ganglia (arrows). Neurons were immunostained for

COX-1 both in normal (A) and inflamed (B) rat colon without appreciable differences in

cytoplasmatic immunostaining. The weak COX-2 signal observed in myenteric neurons from

normal rats (C) was enhanced in neurons and glial cells of myenteric ganglia of DNBS-treated

rats (D). In rats with colitis, COX-2 immunostaining was observed also in muscle cell nuclei

of longitudinal layer (arrowheads).

Figure 4. Representative tracings showing the contractile responses of longitudinal muscle

preparations of colon obtained from normal rats (upper panel) or animals with DNBS-induced

colitis (lower panel). The colonic preparations were incubated in standard medium and

subjected to transmural electrical stimulation (TES: 0.5 ms, 10 Hz, 30 mA, 10 s) either alone

(CON) or in the presence of indomethacin 1 µM (IND), SC-560 0.1 µM or DFU 1 µM. W,

washing; TES, transmural electrical stimulation; CON, control contractions.

Figure 5. Effects of indomethacin (IND, 1 µM), SC-560 0.1 µM, DFU 1 µM and SC-560 plus

DFU on the contractile responses of longitudinal muscle preparations of colon, obtained from

normal rats (A, C) or animals with DNBS-induced colitis (B, D), under the following

conditions: (A, B) incubation in standard medium and application of transmural electrical

stimulation (TES: 0.5 ms, 10 Hz, 30 mA, 10 s); (C, D) incubation in medium containing

guanethidine 10 µM, L-NAME 100 µM, L-732,138 10 µM, GR-159897 1 µM, SB-218795 1

µM, and application of TES. Each column represents the mean ± SEM value obtained from 8-

10 experiments. *P<0.05: significant difference vs control value.


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Figure 6. Upper panel: representative tracings showing the contractile responses of

longitudinal muscle preparations of colon obtained from normal rats or animals with DNBS-

induced colitis. The colonic preparations were incubated in medium containing tetrodotoxin 1

µM and subjected to stimulation with carbachol 1 µM (CARB), either alone (CON) or in the

presence of indomethacin 1 µM (IND). W, washing; CON, control contractions. Lower panel:

column graph showing the effects of indomethacin (IND, 1 µM), SC-560 0.1 µM, DFU 1 µM

and SC-560 plus DFU on the contractile responses of longitudinal muscle preparations

isolated from normal rats and exposed to carbachol 1 µM in the presence of tetrodotoxin 1

µM. Each column represents the mean ± SEM value obtained from 6 experiments.

Figure 7. Effects of indomethacin (IND, 1 µM), SC-560 0.1 µM, DFU 1 µM and SC-560

plus DFU on the contractile responses of longitudinal muscle preparations of colon obtained

from rats with DNBS-induced colitis subjected to subcutaneous injection of superoxide

dismutase 7 mg/kg/day (A) or S-methylisothiourea 14 mg/kg/day (B) for six days. The

colonic preparations were incubated in standard medium and subjected to transmural

electrical stimulation (TES: 0.5 ms, 10 Hz, 30 mA, 10 s). Each column represents the mean ±

SEM value obtained from 10 experiments. *P<0.05: significant difference vs control value.

SOD, superoxide dismutase; SMT, S-methylisothiourea.


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Table 1. Macroscopic and microscopic damage scores estimated for colon in rats under

normal conditions or after treatment with DNBS, DNBS plus SOD or DNBS plus SMT.

Normal DNBS DNBS+SOD DNBS+SMT

Macroscopic

damage

1.56±0.27 10.32±1.44* 6.47±1.60 *,# 5.98±1.15 *,#

Microscopic

damage

1.19±0.31 7.86±1.22* 4.11±1.03 *,# 4.89±1.17 *,#

Each value represents the mean of 8-10 experiments ± SEM. Significant difference from the

respective values obtained in normal rats: *P<0.05. Significant difference from the respective

values obtained in animals treated with DNBS alone: #P<0.05.


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β-actin286 bp

COX-2706 bp

NormalColitis

COX-1882 bp

B

0.0

0.1

0.2

0.3

CO

X-1

/β-a

ctin

(ar

bit

rary

un

its)

A

0.0

0.1

0.2

0.3C *

CO

X-2

/β-a

ctin

(ar

bit

rary

un

its)

Figure 1

Normal Colitis

Normal Colitis

COX-1

COX-2


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100µm

NORMAL COLITIS

A.COX-1 B.COX-1

C.COX-2 D.COX-2

E.NFL F.NFL

G.negative control H.negative control

cm

cm

cm

cm

lmlm

lm lm

lm

lm

lm

lm

cmcm

cmcm

Figure 2


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NORMAL COLITIS

D.COX-2

A.COX-1

C.COX-2

B.COX-1

100µm

Figure 3


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CON

.W

5 mm

2 min.W

.W

.W

.W

.W

.W

.W

.W

.W

.W.W

Figure 4

NORMAL

COLITIS

CONCON

CON CONCON

IND DFUSC-560

IND DFUSC-560

TES TES TES TES TES TES

TES TES TES TES TES TES

JPET #98350This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on February 10, 2006 as DOI: 10.1124/jpet.105.098350

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A. NORMAL

C. NORMAL

B. COLITIS

D. COLITIS

Ch

ang

ein

mot

or

resp

on

ses

toT

ES

(%

)C

han

ge

in m

oto

r re

spo

nse

sto

TE

S (

%)

** *

*

* * *

*

*

*

* **

Figure 5

*

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

0

30

60

90

120

150

180

210

CON IND SC-560 DFU SC-560+DFU





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CON

.W

5 mm

2 min

CARB 1 µM

.W

.W.W

CARB 1 µMCARB 1 µM

CARB 1 µM

INDCON

IND

Ch

ang

ein

mo

tor

resp

on

seto

carb

ach

ol (

%)

0

20

40

60

80

100

120

Figure 6

NORMAL

COLITIS


NORMAL


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Ch

ang

ein

mo

tor

resp

on

seto

TE

S (

%)

A. COLITIS+SOD

Ch

ang

ein

mo

tor

resp

on

seto

TE

S (

%)

*

*

*

*

*

**

*

0

20

40

60

80

100

120

140

160

180

0

20

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60

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100

120

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180

Figure 7

B. COLITIS+SMT




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